The persistence of Neisseria gonorrhoeae, one of the most prevalent bacterial diseases reported in humans, as a major health problem has resulted in the development of numerous methods for detection of Neisseria gonorrhoeae.
Currently accepted procedures for the determination of gonococcal infection rely primarily upon culture techniques. Typical culture techniques include procedures described in Criteria And Techniques For The Diagnosis Of Gonorrhea, published by the Center for Disease Control, Atlanta, Ga. In such culture procedures, a specimen, e.g., a urethral or cervical sample, is placed on an acceptable culture medium, e.g., Thayer-Martin medium. The cultures are incubated at 37.degree. C. in a 5% carbon dioxide atmosphere for 24 to 48 hours. The culture plates are then inspected for the appearance of Neisseria gonorrhoeae colonies. Suspect colonies are gram-stained and tested for oxidase activity. Generally, presumptive diagnosis of gonococcal infection in males is determined by obtaining urethral cultures which exhibit oxidase-positive colonies of gram-negative "coffee-bean" shaped diplococci when cultured on Thayer-Martin medium. In females, gonococcal infection may be diagnosed by examining cervical cultures on Thayer-Martin medium wherein oxidase-positive colonies of gram-negative diplococci appear. Organisms from presumptively identified colonies of Neisseria gonorrhoeae are frequently confirmed by sugar fermentation, fluorescent antibody staining or coagglutination. However, such culture procedures are laborious, time consuming and are generally limited to the detection of "living cells". When culture methods are utilized, a specimen may be taken at one location and shipped to a laboratory, usually at another location where the organisms are cultured and identified. Thus, these culture procedures may require several days before results are obtained. Furthermore, results obtained from culture procedures may be erroneous, if, rather exacting conditions for preservation, shipment, and culturing of the bacteria are not followed.
Nucleic acid hybridization assays have been used as a tool for the detection and identification of a target genetic material such as DNA or RNA. A nucleic acid hybridization assay is premised upon the fact that the target genetic material has a specific nucleotide sequence. It is this sequence of nucleotides that is to be detected. Such detection and identification can be for a specific gene, or DNA or RNA sequence or a point mutation or deletion thereof. A number of techniques exist to carry out such assays. (See Methods In Enzymology, Vol. 68, R. Wu (Ed) pp. 379-469, 1979; and Dunn, A. R., and Sambrook, J., Methods In Enzymology, Vol. 65; Part 1, pp. 468-478, 1980). One of the most widely used procedures is called the Southern blot filter hybridization method (Southern, E., J. Mol. Biol. 98, 503, 1975). This procedure is usually used to identify a particular DNA fragment separated from a mixture of DNA fragments by electrophoretic techniques. The procedure is generally carried out by isolating a sample of DNA from some microorganism. The isolated DNA is subjected to a restriction endonuclease digestion and electrophoresed on a gel (agarose, acrylamide, etc.). When the gel containing the separated DNA fragments is blotted into a suitable matrix, eg. a nitrocellulose filter sheet or diazotized paper, the fragments are transferred and become bound to the matrix. The matrix containing the DNA fragments is then heated to denature the DNA. At this point the matrix is treated with a solution containing a denatured labelled polynucleotide probe and hybridization is allowed to take place. The labelled polynucleotide probe is a nucleotide sequence that is complementary to the DNA fragment that is desired to be detected and which has attached thereto a detectable marker. The detectable marker permits one to verify that the polynucleotide probe has hybridized to the DNA fragment that is desired to be detected. Numerous techniques for labelling a polynucleotide probe with a detectable marker are known. For example, see European Patent Applications publication numbers 0 063 873 and 0 097 373, the disclosures of which are incorporated herein by reference. The unhybridized labelled polynucleotide probe is then separated from the labelled polynucleotide probe that has hybridized to the DNA fragment that is desired to be detected. Separation is generally carried out by washing. The detectable marker of the DNA probe is then detected.
It would be useful to have a polynucleotide probe for the detection of Neisseria gonorrhoeae. A nucleotide sequence derived from the gonococcal cryptic plasmid has been utilized as a polynucleotide probe to detect Neisseria gonorrhoeae. However, such a polynucleotide probe, at best, can only detect those strains of Neisseria gonorrhoeae that contain plasmid DNA. It is believed that from about 40% to about 96%, depending on geographic location, of the strains of Neisseria gonorrhoeae contain plasmid DNA. Since such a polynucleotide probe can only detect those strains of Neisseria gonorrhoeae that contain plasmid DNA, such a polynucleotide probe is of limited utility internationally. See DNA Hybridization Technique for the Detection of Neisseria gonorrhoeae in men with Urethritis, THE JOURNAL OF INFECTIOUS DISEASES, VOL. 148, NO. 3, pages 462-471, September 1983. Therefore, it would be preferred to utilize a nucleotide sequence as a polynucleotide probe that is capable of hybridizing to Neisseria gonorrhoeae chromosomal DNA.
There is an extremely high degree of DNA homology between the chromosomal DNA of Neisseria gonorrhoeae and Neisseria meningitidis, both of which are species of the genus Neisseria. It has been reported that one strain of Neisseria gonorrhoeae and one strain of Neisseria meningitidis have anywhere from about 80% to about 93% chromosomal DNA homology. See Deoxyribonucleic Acid Homologies Amount Species of the Genus Neisseria, Journal of Bacteriology, Vol. 94, No. 4, October 1967, pp. 870-874 and Taxonomy of the Neisseriae; Deoxyribonucleic Acid Base Composition, Interspecific Transformation, and Deoxyribonucleic Acid Hybridization, International Journal of Systematic Bacteriology, Vol. 32, No. 1, January 1982, pp. 57-66. This is an enormously high level of DNA homology, especially in view of the fact that organisms with as little as 70% DNA homology can be considered to be within the same subspecies. See Bergey's Manual of Systematic Bacteriology, Vol. 1, p. 11, published by Williams and Wilkins (1984).
Even further, it is believed that there is an even higher degree of DNA homology between any one strain of Neisseria gonorrhoeae and the sum total of numerous strains of N. meningitidis. This is due to that the portion of the genome of Neisseria gonorrhoeae that is homologous to the chromosomal DNA of each strain of Neisseria meningitidis may not be identical. Consequently, an even smaller percentage, if any, of the Neisseria gonorrhoeae genome is nonhomologous to the sum total of numerous strains of Neisseria meningtidis. A further technical problem is that the portion of the chromosomal Neisseria gonorrhoeae DNA that is nonhomologous to the sum total of numerous strains of Neisseria meningitidis, if any, may not exist as a discrete nucleotide sequence or sequences, but rather, as nucleotide sequences of only a few nucleotides dispersed throughout the Neisseria gonorrhoeae genome. For the purpose of the present invention, a "discrete nucleotide sequence" is a nucleotide sequence greater than about 12 nucleotides.
Moreover, even if a discrete nucleotide sequence of Neisseria gonorrhoeae were to exist that is specific for the strain of Neisseria gonorrhoeae from which it is derived, in order for such sequence to be useful as a polynucleotide probe it is essential that it be specific for other strains of Neisseria gonorrhoeae as well. Otherwise, as with the polynucleotide probe derived from the gonococcal cryptic plasmid, such nucleotide sequence by itself would be of very limited utility.
It should be noted that the genome of any strain of Neisseria gonorrhoeae and Neisseria meningitidis is each about 3 million nucleotides. A skilled scientist can sequence about 2,000 nucleotides per month. Thus, it would take 3,000 scientists one month to sequence the genome of one strain of Neisseria gonorrhoeae and one strain of Neisseria meningitidis.